Biosafety advisory: SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)

Summary of recent changes

  • Classification of full length SARS-CoV-2 RNA as a Risk Group 2 human pathogen and a Risk Group 1 animal pathogen.

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This biosafety advisory is being provided by the Public Health Agency of Canada (PHAC) to assist clinical, diagnostic, and research laboratories in implementing proper biosafety procedures to safely handle samples that may contain SARS-CoV-2. This biosafety advisory also aims to support local risk assessments (LRAs) of facilities where whole live SARS-CoV-2 or purified or chemically synthesized full length SARS-CoV-2 RNA are handled.Footnote 1 SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, which began in December 2019. This biosafety advisory is based on the scientific evidence available as of November 2, 2021, and is subject to review and revisions as new information becomes available.

SARS-CoV-2 is classified as a Risk Group 3 (RG3) human pathogen and as a Risk Group 2 (RG2) animal pathogen. The regulatory oversight of SARS-CoV-2 is under the authority of the PHAC and the Canadian Food Inspection Agency (CFIA). Laboratories receiving specimens from patients that are infected or under investigation for SARS-CoV-2 infection must be aware that improper handling of these specimens poses a risk of exposure, which could seriously impact the health of laboratory personnel and the community, as well as the animal population.

Purified or chemically synthesized full length SARS-CoV-2 RNA is also considered an infectious biological material as it is comprised of single-stranded, positive-sense RNA. Since positive-sense RNA could potentially be translated immediately upon entry into a host cell, purified or chemically synthesized full length SARS-CoV-2 RNA is classified as an RG2 human pathogen and an RG1 animal pathogen. Thus, laboratory procedures that facilitate RNA entry into a cell (e.g., transfection protocols, in vivo work) are subject to increased containment requirements (specified in Section 4.2) to mitigate the risks of exposure and release. SARS-CoV-2 RNA fragments are not considered infectious and are classified as RG1 biological material for both humans and animals.

In this biosafety advisory, SARS-CoV-2 refers to whole live virus, and SARS-CoV-2 RNA refers to purified or chemically synthesized full length RNA.

1.0 Background

The first reports of cases of pneumonia of unknown etiology occurred in the province of Hubei, China in December 2019.Footnote 2 A novel coronavirus, subsequently named SARS-CoV-2, was identified as the causative agent and its genetic sequence was made publicly available to inform the development of diagnostic tests and to support vaccine development.Footnote 2Footnote 3 Commonly reported human COVID-19 symptoms are listed on Coronavirus disease (COVID-19): Symptoms and treatment.Footnote 4 The World Health Organization's (WHO) COVID-19 outbreak website provides information on the number of confirmed cases and deaths.Footnote 5

Some studies have reported that animals (e.g., cats, dogs, minks, lions, tigers) infected with SARS-CoV-2 can develop COVID-19 symptoms including respiratory and digestive symptoms that may result in mortality.Footnote 6Footnote 7Footnote 8 The World Organization for Animal Health (OIE [Office International des Epizooties]), which provides guidance for Veterinary Authorities, has stated that SARS-CoV-2 is an emerging pathogen and that detection in an animal should be reported to the OIE through the relevant national Veterinary Authority, which, for Canada, is the CFIA.Footnote 9 The CFIA must be notified immediately (cfia.notification-notification.acia@inspection.gc.ca) if SARS-CoV-2 is detected in an animal.Footnote 10

2.0 Risk group classification and licensing requirements

SARS-CoV-2 is classified as an RG3 human pathogen and an RG2 animal pathogen. Full length SARS-CoV-2 RNA is classified as an RG2 human pathogen and an RG1 animal pathogen. Controlled activities are defined in Section 7 of the Human Pathogens and Toxins Act (HPTA). Licensing and permit requirements are determined based on sample and activity type, and on whether material containing SARS-CoV-2 or SARS-CoV-2 RNA is imported. More specifically:

For more information on applying for a licence or an import permit, please visit our licensing program web page or contact us at licence-permis@phac-aspc.gc.ca.

3.0 Biosafety recommendations for diagnostic activities

A pathogen in a primary specimen (i.e., in its natural environment) is excluded from the HPTA and is therefore not regulated by the PHAC if the pathogen has not been cultivated or intentionally collected or extracted (e.g., concentrated, cultured). Primary specimens will generally contain lower concentrations of pathogens than found in cultures (i.e., propagated pathogens). Examples of primary specimens include respiratory specimens (e.g., sputum), blood, plasma, feces, and tissues collected directly from patients. Diagnostic specimens from naturally exposed animals (i.e., not resulting from in vivo studies) are also considered primary specimens.

A person who performs laboratory analyses or diagnostic testing that does not involve the cultivation or production of SARS-CoV-2 may be exempt from the licensing requirement as per Section 27(1) of the HPTR. When quality control samples and proficiency panels are needed to perform these tests (e.g., SARS-CoV-2 controls), a person may still be exempt from the licensing requirement based on the activities performed.Footnote 11 A person who performs laboratory analyses that involve identified primary specimens (i.e., primary specimens confirmed to contain SARS-CoV-2) may also be exempt from the licensing requirement based on the type of activities planned (i.e., if they do not cultivate or produce whole live SARS-CoV-2).

When a person performing laboratory analyses or diagnostic activities is exempt from the licensing requirement, it is still recommended that, at minimum, good microbiological laboratory practices be followed in work areas where primary specimens are handled.Footnote 12 All reasonable precautions must be taken to protect the health and safety of the public against the risk posed by these laboratory activities, as per Section 6 of the HPTA. Routine practices and universal precautions are also recommended in laboratories where primary specimens that may contain SARS-CoV-2 are handled.Footnote 13

Examples of diagnostic activities for which routine practices and universal precautions are recommended:

  • clinical chemistry studies, urinalysis, and hematology and serology testing (e.g., analysis with automated platforms)
  • visual examination of inactivated specimens or tissues (e.g., formalin-fixed)
  • visual examination of bacterial and fungal cultures
  • routine staining and microscopic analysis of heat- or chemically-fixed smears
  • assays with virus-inactivated specimens
  • sample preparation for nucleic acid extraction and
  • preparation of specimens for packaging and distribution to diagnostic laboratories for additional testing

If aerosols may be produced during diagnostic activities with primary specimens from patients under investigation for SARS-CoV-2 infection, it is recommended that laboratories also implement additional biosafety recommendations. These recommendations are to be incorporated based on the laboratory's LRA, which takes into consideration the potential for infectious aerosol and droplet production during diagnostic activities and the resulting risk of exposure. As more information regarding SARS-CoV-2 becomes available, it may be determined that there is a lower risk of exposure when handling certain types of primary specimens.Footnote 14 For example, current research with primary specimens from patients under investigation for SARS-CoV-2 infection demonstrates that the viral load in urine poses a low risk of exposure compared to respiratory samples.Footnote 15Footnote 16Footnote 17Footnote 18 Examples of diagnostic activities that may result in the production of aerosols include inoculation of bacterial or fungal culture media, preparation of smears for microscopic analysis, preparation of samples for RT-PCR, and the preparation of frozen sections (unfixed tissues) with a cryostat.

Additional biosafety recommendations

Where a biological safety cabinet (BSC) or other primary containment device is available, the following biosafety recommendations may be implemented:

  • A lab coat, gloves, and face/eye protection are worn when handling primary specimens.
  • Certified BSCs, or other primary containment device (e.g., a closed system, high efficiency particulate air [HEPA] filtered isolators), are used for procedures that may produce infectious aerosols or droplets, and for activities involving open vessels containing infectious material (i.e., not yet inactivated).
  • Centrifugation of primary specimens is carried out in sealed safety cups, or rotors, that are loaded/unloaded in a BSC or other primary containment device (e.g., a closed system, HEPA filtered isolators).

Where a BSC or other primary containment device is not available, the following biosafety recommendations may be implemented:

  • A lab coat, gloves, and face/eye protection are worn when handling primary specimens.
  • Unless other appropriate risk mitigation measures have been implemented in the facility (based on the LRA), respiratory protection that provides a level of filtration of 95% or greater (e.g., N95 respirator) is worn where activities that may potentially generate infectious aerosols or droplets are performed.
  • Centrifugation of primary specimens can be carried out in sealed safety cups or rotors.

Where diagnostic activities are performed outside of a traditional laboratory setting (e.g., point-of-care [POC] molecular testing), risk mitigation measures should be taken to prevent potential exposure to pathogens and the spread of contamination. Depending on the environment, measures could include, but are not limited to:Footnote 19

It is recommended to document any training on the biosafety procedures, as documentation can help demonstrate that all reasonable precautions have been taken to protect the public against the risks posed by these diagnostic activities. Individuals may also refer to the Canadian Biosafety Guideline - Containment Level 1: Physical Design and Operational Practices to identify measures that may help mitigate the risks.Footnote 20

Standard operating procedures

Standard operating procedures (SOPs) may be implemented to reduce the risk of exposure where laboratory activities involve instruments or automated systems that may create aerosols or droplets. For example, these procedures might include inactivating infectious material before it is placed in an automated system (e.g., adding lysis buffer confirmed to inactivate SARS-CoV-2 before samples are placed in an automated system), or opting to use primary containers that are less prone to breakage (e.g., plastic tubes instead of glass tubes).Footnote 21 Laboratories may also consider implementing a waiting period before opening a device to allow sufficient time for potential aerosols or droplets to settle in the event of primary container leaks or breakage (e.g., when using a centrifuge that does not have sealed safety cups or rotors).

To help mitigate the risk associated with downstream processing of infectious material (e.g., by reducing the viral load of a primary specimen), sample preparation protocols that involve SARS-CoV-2 inactivation may be considered.Footnote 22 Many heat-inactivation protocols for coronaviruses (e.g., 56-60°C for 30-60 minutes) have been described in the literature (e.g., for serum and plasma samples), and many sample preparation protocols that inactivate enveloped viruses appear to also effectively inactivate SARS-CoV-2 (e.g., solutions containing Trizol, lysis buffers containing guanidine).Footnote 18Footnote 23Footnote 24 Nevertheless, it is necessary to always validate inactivation procedures in house to confirm their effectiveness against SARS-CoV-2 (i.e., exact conditions, temperature, and duration of incubation). A primary specimen can only be safely handled as a non-infectious sample if the sample preparation protocol has been validated.

As a precautionary measure, disinfection protocols can also be reviewed internally with personnel to ensure that protocols are followed to prevent incidents and injuries. For example, guanidine, which is present in certain solutions and transportation media, has the potential to create a dangerous chemical reaction that releases cyanide gas when exposed to bleach. Thus, it may be necessary to review standard procedures with personnel (e.g., refresher training) to ensure there is no risk of guanidine-rich solutions being exposed to bleach.

Disinfectants

Based on the currently available scientific evidence, chemical disinfectants that are effective against enveloped viruses are effective against SARS-CoV-2. Effective disinfectants include:Footnote 19

For material containing SARS-CoV-2 to be properly inactivated, chemical disinfectants must be used according to the manufacturer's instructions or under published use conditions that have been demonstrated to inactivate enveloped viruses. The use of chemical disinfectants that also denature or inactivate full length SARS-CoV-2 RNA is recommended. The inactivation of infectious material with a chemical disinfectant depends not only on the concentration of the active ingredient, but also on temperature, contact time, and the presence of interfering substances (e.g., organic material). Health Canada provides a list of commercially available hard-surface disinfectants that are likely to be effective against SARS-CoV-2.Footnote 25

4.0 Biosafety requirements for in vitro and in vivo activities

4.1 Biosafety requirements for in vitro and in vivo activities with SARS-CoV-2

SARS-CoV-2 is classified as an RG3 human pathogen and an RG2 animal pathogen. As such, strict containment requirements must be met to reduce the risks of exposure to, and release of, concentrated or propagated material. The following table summarizes the appropriate minimum containment requirements for laboratories where SARS-CoV-2 is intentionally handled. Unless material is excluded from the HPTA, or a person is exempt from the licensing requirement under the HPTR, all in vitro and in vivo activities with SARS-CoV-2 are to be performed in accordance with a Pathogen and Toxin Licence issued under the HPTA and in a facility that meets the minimum applicable containment requirements as summarized in Table 1, and as specified in the CBS.Footnote 26 In vivo activities include experimentally exposing an animal to SARS-CoV-2, and handling these animals and/or their specimens, in a research setting.

Table 1: Canadian containment level requirements for SARS-CoV-2
Activities with SARS-CoV-2 Minimum Containment Level Required

Non-propagative in vitro activities

Examples of these activities include, but are not limited to:

  • preparing human or animal specimens with the goal of concentrating or isolating SARS-CoV-2 for research purposes (e.g., concentration of virus by centrifugation)

CL3

Propagative in vitro activities

Examples of these activities include, but are not limited to:

  • culturing specimens (e.g., propagating the virus)
  • preparatory work for in vivo activities and
  • processing a culture that contains SARS-CoV-2 (i.e., handling propagated or cultivated virus) for packaging and distribution to laboratories

CL3

In vivo work activities

Examples of these activities include, but are not limited to:

  • preparing inoculum
  • inoculating animals and
  • collecting specimens from experimentally infected animals

CL3Footnote 1

Footnotes

Footnote 1

Work in small animal containment zones (SA zones) must meet the applicable requirements in the CL3 column of the CBS and work in large animal containment zones (LA zones) must meet the applicable requirements in the CL3-Ag column of the CBS.

Return to footnote 1 referrer

Many of the CBS requirements are risk- and performance-based and, as such, require that an LRA be performed. Based on the risks associated with in vitro and in vivo activities involving SARS-CoV-2, additional biosafety measures may also be applicable at CL3. Facilities are to conduct an LRA to determine the appropriate risk mitigation measures before undertaking any activity with SARS-CoV-2 (CBS R4.1.8). Furthermore, the LRA and mitigation measures should be updated as new information becomes available.

4.2 Biosafety requirements for in vitro and in vivo activities with full length SARS-CoV-2 RNA

Full length SARS-CoV-2 RNA is classified as an RG2 human pathogen and an RG1 animal pathogen. As such, certain containment requirements must be met to reduce the risks of exposure to, and release of, infectious material. Unless material is excluded from the HPTA, or a person is exempt from the licensing requirement under the HPTR, all in vitro and in vivo activities with purified or chemically synthesized full length SARS-CoV-2 RNA are to be performed in accordance with a Pathogen and Toxin Licence issued under the HPTA, and in a facility that meets the minimum applicable containment requirements as summarized in Table 2, and as specified in the CBS.Footnote 26 In vivo activities include experimentally exposing an animal to full length SARS-CoV-2 RNA, and handling these animals and/or their specimens, in a research setting.

Table 2. Canadian containment level requirements for full length SARS-CoV-2 RNA
Activities with full length SARS-CoV-2 RNA Minimum Containment Level Required

In vitro activities that are unlikely to result in entry of SARS-CoV-2 RNA into a cell

Examples of these activities include, but are not limited to:

  • analyzing or modifying the full length genomic sequence
CL2Footnote 1

In vitro activities that may result in entry of SARS-CoV-2 RNA into a cell

Examples of these activities include, but are not limited to:

  • transfecting an immortalized cell line with full length SARS-CoV-2 RNA
CL3

In vivo work activities involving SARS-CoV-2 RNA

Examples of these activities include, but are not limited to:

  • injecting full length SARS-CoV-2 RNA into an animal
  • collecting specimens from experimentally infected animals
CL3Footnote 2

Footnotes

Footnote 1

In accordance with the conditions of the RG2 Pathogen and Toxin Licence, certain in vitro activities listed in the licence may be permitted at CL1 if laboratories follow the biosafety recommendations described in this biosafety advisory.

Return to footnote 1 referrer

Footnote 2

Work in small animal containment zones (SA zones) must meet the applicable requirements in the CL3 column of the CBS, whereas work in large animal containment zones (LA zones) must meet the applicable requirements in the CL3-Ag column of the CBS.

Return to footnote 2 referrer

Should new information result in changes to the risk group classifications of SARS-CoV-2 or full length SARS-CoV-2 RNA, or to this biosafety advisory, or result in the publication of a biosafety directive, such information will be reflected in the ePATHogen - Risk Group Database.Footnote 27

5.0 Transportation

The transportation of SARS-CoV-2 is subject to the Transportation of Dangerous Goods Regulations (TDG Regulations) and must meet the packaging requirements stipulated in the standard CAN/CGSB-43.125. Packaging requirements are based on the proper classification of the material, as summarized in the box below. Materials that are not considered infectious as per the TDG Regulations (e.g., SARS-CoV-2 RNA) or that are exempt from the packaging requirements of the TDG Regulations are still required to meet normal transportation conditions, such as leak-proof packaging.

Classification of material potentially contaminated by SARS-CoV-2

Please consult the TDG Regulations if other infectious substances/dangerous goods are present.

SARS-CoV-2 cultures are always assigned to UN2814, Infectious substance, affecting humans, Class 6.2, Category A.

SARS-CoV-2 samples that are in a form other than a culture are assigned to one of the following classifications:

  • UN2814, Infectious substance, affecting humans, Class 6.2, Category A or
  • UN3373, Biological substance, Category B, Class 6.2, Category B, in accordance with the conditions set out in paragraphs 1.39(a) to (c) of the TDG Regulations

Medical or clinical waste related to SARS-CoV-2 is assigned to:

  • UN2814, if it contains Category A infectious substances, such as propagated SARS-CoV-2
  • UN3291, if it contains Category B infectious substances or if there are reasonable grounds to believe that it has a low probability of containing infectious substances such as SARS-CoV-2. Such waste must be identified as "Clinical waste, unspecified, n.o.s.", "Biomedical waste, n.o.s", "Medical waste, n.o.s.", or "Regulated medical waste, n.o.s."
Dried blood spots collected by applying a drop of blood onto absorbent material are not subject to the TDG Regulations. Environmental samples (e.g., food, water, wastewater) that do not pose a significant risk of infection are not subject to the requirements for infectious substances of Class 6.2 under the TDG Regulations However, whereas dried blood spots and environmental samples do not need to meet the packaging requirements of the TDG Regulations, these samples still need to meet certain packaging requirements to ensure safe transport conditions and protect the quality of the sample, to obtain reliable and accurate diagnostic results.

Primary specimens from asymptomatic individuals who are not known to be positive for an infectious disease such as COVID-19 can be shipped as "exempt human specimens", following the conditions of Exemption 1.42 of the TDG Regulations. Please note that precautionary measures may be required, based on factors such as known medical history, symptoms, individual circumstances, and endemic local conditions. For example, primary specimens from asymptomatic babies of mothers infected with SARS-CoV-2 or that are presenting COVID-19 symptoms (i.e., diagnosed or undiagnosed) could be classified as Category B infectious substances based on the likelihood of contamination.

For more information, consult the Transport Canada Transportation of Dangerous Goods website or the Transport Canada Shipping Infectious Substances bulletin. To obtain further assistance, contact Transport Canada at:

In the event of an emergency involving dangerous goods, call CANUTEC at 1-888-CANUTEC (226-8832), 613-996-6666 or *666 on a cellular phone.

6.0 Contact information

Further biosafety information may be obtained by visiting the PHAC Centre for Biosecurity website or contacting us by:

To notify the CFIA that SARS-CoV-2 has been detected in an animal, or to receive more information regarding reportable and immediately notifiable diseases in animals, contact the CFIA by email at cfia.notification-notification.acia@inspection.gc.ca.

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